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. 2021 Mar 8;118(11):e2010206118. doi: 10.1073/pnas.2010206118

Fig. 6.

Fig. 6.

JAK/STAT inhibitors block IFN activation, proliferation, and migration in CAV1-deficient primary human PAECs but fail to reduce constitutive AKT activation. (A) CXCL10 protein was reduced by baricitinib (100 nM), ruxolitinib (100 nM), or tofacitinib (100 nM) in both CAV1-silenced and siCTRL treated PAECs. After siRNA transfection (48 h), PAECs were serum-starved for 24 h followed by 24 h of drug treatment or vehicle control prior to cell supernatant collection. CXCL10 concentration, measured by ELISA, presented as mean ± SE. (B) Treatment with baricitinib (100 nM), ruxolitinib (100 nM), and tofacitinib (100 nM) all reduced cell proliferation, as assessed by bromodeoxyuridine (BrdU) incorporation, in CAV1-silenced PAECs. BrdU incorporation was unaffected in siCTRL transfected PAECs treated with either baricitinib, ruxolitinib, or tofacitinib (P ≥ 0.20 for all compared to vehicle-treated siCTRL cells). After CAV1 knockdown (48 h), PAECs were transferred to 96-well plates in complete media for 6 h, then serum-starved for 24 h and returned to complete media with the specified JAK/STAT inhibitor or vehicle control. BrdU was added after 48 h of drug treatment and incorporation was assessed 24 h later. Data presented as mean FC ± SE. (C) Treatment with baricitinib (100 nM), ruxolitinib (100 nM), or tofacitinib (100 nM) significantly reduced cell migration in CAV1-silenced PAECs (P = 0.02 for the interaction between CAV1-silencing and drug treatment). After CAV1 knockdown (48 h), PAECs were transferred to 96-well plates containing a circular gel insert (Oris Pro Cell Migration Assay) for 6 h, then serum-starved for 24 h, followed by return to complete media with the specified JAK/STAT inhibitor or vehicle control and the insert was removed. Percent closure of the monolayer defect was assessed 48 h later. Data presented as the mean ± SE with representative images at 0 and 48 h shown (Right; 10× magnification). Replication throughout represent independent experiments each using different PAEC donors. *P < 0.05, **P < 0.01, ***P < 0.001.